Members of the phospholipase C family of enzymes are key mediators of cellular signal transduction. Many extracellular signaling molecules including hormones, growth factors, neurotransmitters, and immunoglobulins bind to their respective cell surface receptors and activate phospholipase C enzymes. In general, biological systems that are activated by receptor tyrosine kinase function in part by effecting the activation of an phospholipase-C enzyme. Activated phospholipase C then catalyzes the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2), a component of the plasma membrane, to produce diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
In their respective biochemical pathways, IP3 and diacylglycerol serve as second messengers that trigger a series of intracellular responses. IP3 induces the release of calcium from intracellular stores, and diacylglycerol activates protein kinase C (PKC). The calcium ions directly regulate target enzymes and indirectly affect other enzymes by functioning as a second messenger and interacting with calcium-binding proteins, such as troponin C and calmodulin. For example, calcium ions regulate muscle contraction, glycogen breakdown and exocytosis. Diacylglycerol, a product of the hydrolysis by PLCs, acts as a second messenger by activating protein kinase C. Activated protein kinase C phosphorylates a number of intracellular proteins at the serine and threonine residues to thereby regulate downstream intracellular signalling. For example, the phosphorylation of glycogen synthase by protein kinase C inhibits the synthesis of glycogen. Protein kinase C can also control cell division and proliferation.
Both the calcium and the PKC pathways are major branches of transmembrane signal transduction circuits, which regulate numerous cellular processes, including secretion, neural activity, metabolism, and proliferation. Hence, phospholipase C molecules have many regulatory functions including: regulation of glycogenolysis in liver cells, histamine secretion by mast cells, serotonin release by blood platelets, aggregation by blood platelets, insulin release by pancreatic islet cells, epinephrine secretion by adrenal chromaffin cells, and smooth muscle contraction. Evidence indicates that a high percentage of primary human mammary carcinomas concomitantly show abnormally high levels of PLC-gamma-1 (Kassis et al., Clin Cancer Res., Aug;5(8):2251-60, 1999). Likewise, studies on spontaneous hypertensive rats have suggested that one of the main causes for the hypertension is an abnormal activation of PLC-delta-1 resulting from point mutations in the X and Y regions of the PLC amino acid sequence (Sanada et al., Hypertension 33(4):1036-42, 1999).
Phospholipase C molecules have been found in a broad spectrum of organisms including bacteria, simple eukaryotes, plants and animals (Munnik et al.(1998) Biochim. Biophys. Acta. 1389:222-272,). Several distinct isoforms of phospholipase C have been identified in animals and are categorized as PLC-xcex2, PLC-xcex3, and PLC-xcex4. Subtypes are designated by adding Arabic numbers after the Greek letters, e.g., PLC-xcex21. PLCs have a molecular mass of 62-68 kDa, and their amino acid sequences show two regions of significant similarity.
The present invention is based, in part, on the discovery of a novel phospholipase C family member, referred to herein as xe2x80x9c16835xe2x80x9d. The nucleotide sequence of a cDNA encoding 16835 is shown in SEQ ID NO:1, and the amino acid sequence of a 16835 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3.
Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 16835 protein or polypeptide, e.g., a biologically active portion of the 16835 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 16835 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3. or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865, wherein the nucleic acid encodes a full length 16835 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 16835 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 16835 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 16835 nucleic acid molecules and polypeptides.
In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 16835-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 16835 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 16835 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 16835-mediated or -related disorders. In another embodiment, the invention provides 16835 polypeptides having a 16835 activity. Preferred polypeptides are 16835 proteins including at least one phospholipase C domain (e.g., a PLC-X domain (from about amino acids 285-430 of SEQ ID NO:2) and a PLC-Y domain (from about amino acids 474-591 of SEQ ID NO:2)), a calcium binding (C2) domain (from about amino acids 609-699 of SEQ ID NO:2), and/or a pleckstrin homology (PH) domain (from about amino acids 11-119 of SEQ ID NO:2), and, preferably, having a 16835 activity, e.g., an activity as described herein, e.g., the ability to catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) producing diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
In other embodiments, the invention provides 16835 polypeptides, e.g., a 16835 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Numbers 1864 and 1865, wherein the nucleic acid encodes a full length 16835 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs which include a 16835 nucleic acid molecule described herein.
In a related aspect, the invention provides 16835 polypeptides or fragments operatively linked to non-16835 polypeptides to form fusion proteins. In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 16835 polypeptides.
In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 16835 polypeptides or nucleic acids.
In still another aspect, the invention provides a process for modulating 16835 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 16835 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation.
In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of a 16835-expressing cell, in a subject. Preferably, the method includes comprising administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 16835 polypeptide or nucleic acid.
In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition. Most preferably, the disorder is a cancer, e.g., a solid tumor, a soft tissue tumor, or a metastatic lesion. Preferably, the cancer is a sarcoma, a carcinoma, or an adenocarcinoma. Preferably, the cancer is found in a tissue where a 16835 polypeptide or nucleic acid is expressed, e.g., colon, breast, ovarian, or liver cancer. Most preferably, the cancer is found in the breast, ovary, colon, liver and lung.
In a preferred embodiment, the compound is an inhibitor of a 16835 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). The inhibitor can also be a phospholipase inhibitor or a derivative thereof, or a phosphoinositol analog.
In a preferred embodiment, the compound is an inhibitor of a 16835 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.
In a preferred embodiment, the compound is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.
The invention also provides assays for determining the activity of or the presence or absence of 16835 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis. Preferably, the biological sample includes a cancerous or pre-cancerous cell or tissue. For example, the cancerous tissue can be a solid tumor, a soft tissue tumor, or a metastatic lesion. Preferably, the cancerous tissue is a sarcoma, a carcinoma, or an adenocarcinoma. Preferably, the cancerous tissue is from the breast, ovarian, colon, lung, liver, kidney, or brain.
In a further aspect the invention provides assays for determining the presence or absence of a genetic alteration in a 16835 polypeptide or nucleic acid molecule in a sample, for, e.g., disease diagnosis. Preferably, the sample includes a cancer cell or tissue. For example, the cancer can be a solid tumor, a soft tissue tumor, or a metastatic lesion. Preferably, the cancer is a sarcoma, a carcinoma, or an adenocarcinoma. Preferably, the cancer is a breast, ovarian, colon, lung, liver, kidney, or brain cancer.
In a still further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., proliferative disorder, e.g., cancer (e.g., breast, ovarian, colon, liver or lung cancer). The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 16835 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 16835 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder.
In a preferred embodiment, the disorder is a cancer of the breast, ovary, colon, or liver. The level of 16835 nucleic acid or polypeptide expression can be detected by any method described herein.
In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 16835 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.
In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 16835 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 16835 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 16835 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous tissue where a 16835 polypeptide or nucleic acid is obtained, e.g., a cancer of the breast, ovary, colon, lung, or liver.
In a preferred embodiment, the sample is a tissue sample (e.g., a biopsy), a bodily fluid, cultured cells (e.g., a tumor cell line).
The invention also provides assays for determining the activity of or the presence or absence of 16835 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 16835 polypeptide or nucleic acid molecule, including for disease diagnosis.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 16835 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 16835 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 16835 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.